The regulated release of proteins from dense-core granules (DCGs) is critical to fundamental processes such as neuronal survival and synaptic transmission. Many of the underlying molecular events that drive the synaptic secretion of proteins from DCGs have not been elucidated. Studies in this proposal are directed at characterizing the mechanisms that facilitate the synaptic secretion of an important regulated secretory protein, tissue-type plasminogen activator (tPA), from DCGs in hippocampal neurons. tPA is a serine protease that is implicated in the modification of synaptic efficacy during learning and memory. tPA modulates glutamatergic transmission by interacting with the N-methyl-D-aspartate (NMDA) receptor and influences synaptic plasticity by promoting the cleavage of another secreted neuromodulatory protein, brain derived neurotrophic factor (BDNF). tPA and other key neuromodulatory proteins such as BDNF are released from postsynaptic sites in an activity-dependent manner. However, limited insight exists regarding the molecular machinery that mediates release of these proteins from postsynaptic sites. In Specific Aim 1, experiments are proposed to assess the relevance of a recently identified exocytic microdomain in dendritic spines to the secretion of neuromodulatory proteins from DCGs. The proposed experiments are directed at discerning if postsynaptic DCG exocytosis is spatially restricted to this syntaxin-4 enriched exocytic domain and if syntaxin-4 is required for DCG exocytosis. Following the postsynaptic release of neuromodulatory proteins, synaptic pools of these proteins must be replenished. In Specific Aim 2, experiments are proposed to assess if synaptic capture occurs at postsynaptic sites subsequent to activity-dependent exocytosis. These experiments rely on the use of a tPA-photoactivatable fluorescent protein hybrid and a photoactivation-chase approach to track movement of tPA-containing DCGs after neuronal stimulation. If stimulation is found to trigger synaptic capture of transiting DCGs, the mechanistic requirements for synaptic capture will be examined. tPA-containing DCGs localize to both pre- and postsynaptic sites in hippocampal neurons. While release of tPA from postsynaptic sites has been characterized, the requirements for presynaptic release of tPA have not been systematically evaluated. In Specific Aim 3, experiments are proposed to discern how patterned electrical stimulation protocols such as those known to be effective at inducing LTP and LTD influence the release of tPA from presynaptic sites. These studies will contribute to an understanding of the cellular determinants that favor either pre- or postsynaptic secretion of DCGs. Findings derived from these studies are relevant not only to physiological processes in the nervous system such as learning and memory but also to neuropathological processes. Excitotoxic cell death and the neuronal degeneration associated with Alzheimer<s disease are linked to tPA-mediated proteolysis. PUBLIC HEALTH RELEVANCE: The proposed studies focus on the cellular mechanisms that regulate the synaptic secretion of tissue-type plasminogen activator (tPA) from hippocampal neurons. Regulated release of this enzyme modulates physiological functions such as memory-related plasticity. tPA-mediated extracellular proteolysis is also implicated in excitotoxic cell death and the neuronal degeneration associated with Alzheimer<s disease.